Xin Zhao

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

ABSTRACT Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

Defense of the Bovine Mammary Gland by Polymorphonuclear Neutrophil Leukocytes

The primary phagocytic cells of the bovine mammary gland, polymorphonuclear neutrophil leukocytes (PMN), and macrophages, comprise the first line of defense against invading pathogens. In the normal healthy mammary gland, macrophages predominate and act as sentinels to invading mastitis-causing pathogens. Once invaders are detected, macrophages, and possibly mammary epithelial cells, release chemoattractants that direct migration of PMN into the area. In the mammary gland, protection is only effective if rapid influx of PMN from the circulation and subsequent phagocytosis and killing of bacteria occur. The second line of defense against infection consists of a network of memory cells and immunoglobulins that interact with the first line of defense. To minimize mammary tissue damage caused by bacterial toxins and oxidative products released by PMN, elimination of invading bacteria must proceed quickly. Therefore, the inflammatory response needs to be regulated. Hormones, metabolites, and acute phase proteins act to influence the outcome of mastitis, especially around parturition. The number of circulating PMN in cows during early lactation is highly heritable and closely related to susceptibility to clinical mastitis at this time. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host–pathogen interactions. For example, the cloning and expression of proteins such as recombinant bovine soluble (rbos) CD (cluster of differentiation) 14 antigens, may provide ways of minimizing damaging effects of endotoxin during acute coliform mastitis. Soluble CD14 binds and neutralizes lipopolysacharide (LPS) and causes local recruitment of PMN after binding of CD14-LPS complexes to mammary epithelial cells. Development of transgenic animals that express rbosCD14 in their milk could prevent infection by Gram-negative pathogens.

Effects of diets containing different concentrations of mannanoligosaccharide or antibiotics on growth performance, intestinal development, cecal and litter microbial populations, and carcass parameters of broilers

The effects of 2 levels of mannanoligosaccharide (MOS) in feed were compared with antibiotic growth promoters on growth performance, intestinal morphology, cecal and litter microbial populations, and carcass parameters in broilers raised in a sanitary environment. Dietary treatments included: 1) antibiotic growth promoter-free diet (control), 2) VIRG (diet 1 + 16.5 mg/kg of virginiamycin), 3) BACT (diet 1 + 55 mg/kg of bacitracin), 4) LMOS (diet 1 + 0.2% MOS), and 5) HMOS (diet 1 + 0.5% MOS). Birds were randomly assigned to 3 replicate pens/treatment (n = 55/pen). Body weight and feed intake were recorded weekly throughout 38 d. At d 14, 24, and 34, a 1-cm segment of duodenum, jejunum, and ileum was used in morphological analysis (n = 9 birds/d per treatment). At the same bird ages, cecal contents were assayed for lactobacilli, bifidobacteria, Salmonella, Campylobacter, and Escherichia coli, whereas litter was analyzed for Salmonella, Campylobacter, and E. coli. Carcass yields (breast fillet and tenders, thigh, drumstick, and wing) were determined at d 38. Body weight, feed conversion, and carcass yields did not differ among treatments. In contrast to birds fed VIRG or BACT, LMOS and HMOS consistently increased (P < 0.05) villi height and goblet cell number per villus in all intestinal segments at d 24 and 34. Bifidobacteria concentrations were higher (P < 0.05) in LMOS- and HMOS-fed birds at all time points. Birds and litter from all treatments were free of Salmonella. At d 14 and 24, cecal E. coli and Campylobacter counts were not different among treatments. In comparison to birds fed control, at d 34, BACT, LMOS, and HMOS significantly reduced (P < 0.05) cecal E. coli concentrations, whereas Campylobacter counts were reduced (P < 0.05) by VIRG, BACT, and LMOS. Litter bacterial counts were not altered by dietary treatments. In conclusion, under conditions of this study, MOS conferred intestinal health benefits to chickens by improving its morphological development and microbial ecology. But, there were no additional benefits of the higher MOS dosage.

A critical analysis of disease-associated DNA polymorphisms in the genes of cattle, goat, sheep, and pig

Genetic variations through their effects on gene expression and protein function underlie disease susceptibility in farm animal species. The variations are in the form of single nucleotide polymorphisms, deletions/insertions of nucleotides or whole genes, gene or whole chromosomal rearrangements, gene duplications, and copy number polymorphisms or variants. They exert varying degrees of effects on gene action, such as substitution of an amino acid for another, shift in reading frame and premature termination of translation, and complete deletion of entire exon(s) or gene(s) in diseased individuals. These factors influence gene function by affecting mRNA splicing pattern or by altering/eliminating protein function. Elucidating the genetic bases of diseases under the control of many genes is very challenging, and it is compounded by several factors, including host × pathogen × environment interactions. In this review, the genetic variations that underlie several diseases of livestock (under monogenic and polygenic control) are analyzed. Also, factors hampering research efforts toward identification of genetic influences on animal disease identification and control are highlighted. A better understanding of the factors analyzed could be better harnessed to effectively identify and control, genetically, livestock diseases. Finally, genetic control of animal diseases can reduce the costs associated with diseases, improve animal welfare, and provide healthy animal products to consumers, and should be given more attention.

Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine – a review

Abstract Determining the structure of the intestinal receptor for enterotoxigenic Escherichia coli (ETEC) K88 fimbriae will make it possible to develop new strategies to prevent K88+ ETEC-induced disease in pigs. Putative K88 adhesin receptors have been identified in both intestinal brush border and mucus preparations as either glycoproteins or glycolipids. Proteins with sizes of 25, 35, 40–42, 60, and 80 kDa in the intestinal mucus and 16, 23, 35, 40–70, 74, 210, and 240 kDa in brush border membranes were reported to bind specifically to K88ab and K88ac fimbriae. The factors accounting for these variable results may include the variants of K88, ages, breeds, and phenotypes of pigs, and even the sampling sites in the small intestine. Of the reported K88 receptors, only three brush border receptors, i.e., a pair of mucin-type sialoglycoproteins (210 kDa or 240 kDa), an intestinal neutral glycosphingolipid (IGLad), and a 74-kDa transferrin glycoprotein (GP74), have fulfilled the criteria as phenotype-specific K88 fimbrial receptors. Inhibiting the attachment of ETEC to intestine by modifying the receptor attachment sites has been the key for developing novel approaches to preventing ETEC-induced diarrhea in pigs. These include: (1) receptor analogs from a variety of biological sources, (2) an enteric protected protease, (3) chicken egg-yolk containing anti-K88 fimbrial antibodies, and (4) some Lactobacillus isolates producing proteinaceous components or carbohydrates interacting with mucus components. Future studies should be directed to further characterize the carbohydrate and protein moieties of receptors recognized by the K88 adhesin variants and to identify the genes responsible for susceptibility to K88+ infections.

Effects of Galacto-Oligosaccharides and a Bifidobacteria lactis-Based Probiotic Strain on the Growth Performance and Fecal Microflora of Broiler Chickens

A galacto-oligosaccharide (GOS) prebiotic was prepared by reacting a high concentration of lactose (40% wt/vol) with a beta-galactosidase enzyme for 24 h at 37 degrees C. The enzyme was produced from recombinant Pichia pastoris X-33 cells. The study aimed at evaluating the effects of the prebiotic, a Bifidobacterium lactis-based probiotic, and the combination of these dietary additives on BW, feed intake, feed conversion ratio, and fecal counts of total anaerobic bacteria, lactobacilli, and bifidobacteria in broiler chickens. No significant differences in BW, feed intake and feed conversion ratio were found among the various groups. The study showed that GOS selectively stimulated the fecal microflora of broiler chickens. Total anaerobic bacteria and lactobacilli were increased by 3.4- and 3.56-fold, respectively, in chickens fed the diet containing GOS (3 kg per 25 kg) and B. lactis for 40 d compared with those fed the control diet. The bifidobacteria population in chickens fed the diet containing GOS (3 kg per 25 kg) and B. lactis significantly increased 21-fold in comparison to the control-fed birds. In particular, increasing the dietary concentration of GOS was accompanied by significant increases (P < 0.05) in bifidobacteria counts. The detectable population of bifidobacteria was also greater (P < 0.05) in chickens fed the diet containing GOS and bifidobacteria when compared with chickens fed a bifidobacteria-containing ration only. These results suggest that using GOS in combination with a B. lactis-based probiotic favored intestinal growth of bifidobacteria in broiler chickens.

Escherichia coli induces apoptosis and proliferation of mammary cells.

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1β converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation. Cell Death and Differentiation (2001) 8, 808–816

Recombinant Soluble CD14 Reduces Severity of Intramammary Infection by Escherichia coli

ABSTRACT The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 μg of LPS, 0.3 μg of LPS plus 100 μg of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 μg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.

Bovine interleukin-1 expression by cultured mammary epithelial cells (MAC-T) and its involvement in the release of MAC-T derived interleukin-8.

MAC-T cells, an established bovine mammary epithelial cell line, were utilized to investigate both expression of interleukin-1 (IL-1) mRNA and secretion of IL-1 after Escherichia coli lipopolysaccharide (E. coli LPS) stimulation. In addition, recombinant human IL-1beta, recombinant human IL-1 receptor antagonist (IL-1ra) and a neutralizing goat antibody against type I human IL-1 receptor were used to study the involvement of IL-1 in the release of IL-8. The expression of MAC-T derived IL-1alpha mRNA was correlated to production of IL-1alpha protein as measured by an IL-1alpha sandwich ELISA. Secretion of IL-1alpha was dose- and time-dependent, with a maximal level of 600 pg/ml detectable upon 2-h stimulation with 20 microg of LPS per ml. IL-1ra and the neutralizing antibody significantly blocked the ability of IL-1beta to stimulate secretion of IL-8 by MAC-T cells. During this study, we have demonstrated that MAC-T cells secrete IL-1 in response to LPS stimulation and IL-1 is an important mediator for the release of the bovine IL-8 by MAC-T cells. These results further indicate the potential importance of mammary epithelial cells as a source of immunoregulation in the mammary gland via cytokine elaboration.

Lipases from Mammals and Fishes

Lipases are a broad family of enzymes that catalyze the hydrolysis of ester bonds in substrates such as triglycerides, phospholipids, cholesteryl esters, and vitamin esters. Lipases are receiving increasing interest due to their effects on the quality of food products (e.g., the quality of post-harvest seafoods), and their actual and potential applications in modified foods and industrial processes. Lipases that catalyze specific reactions and that are active at particular conditions of pH and temperature to suit the requirements of industrial processes are of particular interest. This review focuses on lipases that display predominantly triacylglycerol hydrolase activity. Section 1 presents an overview of lipases from terrestrial organisms and the lipase gene family members. Due to their unique physiology, diet, and habitat, fish lipases may demonstrate novel activities that have potential applications for bioindustrial catalysis. Section 2 discusses lipases from fish and compares them with lipases from mammals and birds. Purification strategies and properties of the isolated enzymes are reviewed in detail.